Process for extracting vegetable protein



, oleaginous,

' the efficiency of the process.

found that calcium bromide, calcium hydroxide.

Y 3 protein being substantially free of mucilage, other carbohydrates,land color components. t.

Other .obj ects'of this invention willbecome apparent Vvupon a morecomplete understanding of the invention as hereinafter set forth. ,V

These objects, as well as others that willl be apparent hereinafter, maybe accomplished by the s The concentrations of sodium hydroxide solutionand calcium chloride solutionshould preferably besuch that whenadmi'xed,as previously described, the normality of the sodium" hydroxide is about0.5 and the normality of the calcium chloridexis between about 0.1 andabout 0.6. As

present invention by admixing a comminuted," proteinaceous vegetablemeal, preferablyv one which has been previously oilzextracted, andanaqueous solution prepared by admixing sodiumf gthe concentration-of thecalcium chloride is increased, the carbohydrate and pigment content pf;the Protein product decreases. At a calcium chloride concentrationbeyond about 0.6 N.. no

' furtherJimprovement in product quality is obhydroxide, calciumchloride, and `water.` Y The Y mixture is vigorously stirred andthesolids re"k moved from the solution. The resulting solution is thenacidied, and the precipitated-protein is separated from the solution,Washed, and dried.

YIt has been found that the above process is applicable to suchvegetable meals as those'ob- .Y tained from voil extracted peanuts,soybeans, flaxseed, cottonseed, and castor beans. The termsproteinaceous vegetable residue and, Vegetable residue; or foilseedmeal, as previously and hereinafter used in the description and claims,refer to vegetable matter suchA as the aforementioned seeds and beans aswell as Yothers of similar character from which substantial` Y ofcalcium chloride and sodium hydroxide. The Y solution is preferablyprepared by adding, with y rapid stirring, an aqueous solution of sodiumhydroxide to an aqueous solution of calcium chloride. 'I'he calciumhydroxidethus produced is believed to serve as avery eiicient medium forthe remove-lof. pigment components and carbohydrates. Thischaracteristic of the calcium hydroxide thus produced in situ is-thoughtto be due to the adsorptive properties of the compound in the negranularform produced by this method. It has been found that if thereverse-is done (that is, the calcium chloride added to the sodiumhydroxide), relativelyr coarse particles of calcium hydroxide are formedwhich greatly impair It has also been calcium oxide, and otherwater-soluble calcium compounds may be used, although they are far lessefficient when substituted for calcium chloride in the instant novelprocess. It is possible,

through the use ofcalcium chloride -in the .in-

stant novel process, to obtain a protein substantially free ofcarbohydrates and pigmentV components. Likewise, if desired, a protein`containing more pigment component may be produced by simply varying theconcentration of the cal-A cium chloride in the solution.

The normalities of the calcium chlo-ride Vsolutions used werei'lguredonthe basis of anhydrous calcium chloride {ho-Wever, it is to beunderstoodwater of crystallization may be employed `Vwith `equally good results,andthat there is no intentionV to limit the invention to thev anhydrousvariety of calcium chloride. e

served, although the product is substantially free 'ofthe aforementionedcarbohydrates and pig- Vments. The aforementioned calciumchloridesodiumhydroxide solution generally may be prepared by pouring,with rapid stirring, one volume of `1.0 N. sodium hydroxide about anequal 4volumepf an aqueous solution,0fcalcium chloridewhoseconcentration isA twice the .normality 20.

of the concentration of calcium chloride desired in the nalaforementioned solution. oleaginous, proteinaceous vegetable residue,from which substantial amounts of oil have been removed .by solventextraction or other suitable means, is then admixed with theaforementioned solution of calcium chloride'and sodium hydroxide,preferably inthe ratio of about 3 grams of vegetable residue to about100 cc. ofextracting solution. This ratio may be varied to some degreedepending upon the type of oilseed used and the quality of the 'productdesired. The mixture is vigorously shaken, or otherwise stirred, for aperiod of about two hours. At the end of this period of mixing, which ispreferably carried out at room temperature, the solids are removed fromthe mixture by centrifuging, filtering, or other means, and theresulting slightly turbid solution is adjusted to api-I of betweenabout,3.5 and about-,4.5 depending vuponfthe vegetable seed meal used asa starting material. Any acidic material, nonoxidizing under theconditions obtaining, which will give the desired pH may be used;economic considerations being the primary limitation. Thus, whileconcentrated hydrochloric acid is preferred, dilute sulfuric acid oracetic acid, for example, may also `be employed. i

Upon the addition vof acid, with stirring, the

`proteins rapidly precipitate and generally may' be separated from th'emother liquor about one-half hour afterl -acidication After separation,the

i protein is Washed with an'acidiedaqueous'solu- A tion of about 5%sodiumchloride.A This'washing ever, the vproduct is soluble 1 v --tionsof the presentinvention.- that other forms of calcium chloridecontainingfp.

rremoves substantially' all of Y the vpigmented mother liquor adheringto the proteins. The product is ,then driedin any -suitable manner, forexample'in vacuo, at between about 45" and about 55 C. If 0.5 N. sodiumhydroxide aqueous solution is used instead of the aforementioned aqueouscalcium chloride and sodium hydroxide solution, a product isobtained'which has as much as 7.8% ,byv weight of carbohydrateimpurities and is strongly pigmented. In either case, howin dilutealkaline solution.

For a, clearer understanding of the instant novel process, reference maybe had to the examples and tables which are hereinafter set forth.

, It is to be understood, however, that these tables and examples are.nottobeconstrued as limita- '(solvent extracted) ilaxseed meal wasadded, at

@9er-.temperaturen solution preparedeby addingvinti-1fstwngaboutf()fcc:'-ofaboutl'N.4 sodium hydroxide' Y1to1 about anequal'lvolume of 1 about lilfNfaqueoiis ealcium''chloride. Afterafper-i'od of shaking forJ about tw'o hoursfthe solution was filtered,and the resulting slightly turbid filtrate was acidied with concentratedhydrochloric acid to a pH of about 3.75. The precipitate was washed withstirring for about one-half hollrwithv about 500 cc. of about 5% aqueoussodiumchlo.- e

ride adjusted to a pH of about 3.75 Withfhfydrochloric acid. Filteringvyielded a protein 'which when dried in` vacuo at not above 50 Cwasalmost pure white and amounted to a littleioverA grams dry weight.

Table I illustrates the properties and purity' -lwith dilutehydrochloric iacid. The resulting mixture Wasstirred for vabout tenminutes toy break .11p thecl'umps land remove the adhered,

. lslightly colored mother liquor and centrifuged of the proteinisolated from a substantially ciliegi again; The.proteins.thus.treatedlweradried-um :der infra red light and yieldedslightly morathan .2-'Zf ;grams dry Weight of a--substantially whitetheLprocedure-set `fcrth. in 'Example' I was em- .plcyedtoisolate theprotein. "However, asindif-A .catedincolumn 1 .ofTableL the normalities'of vthe components of' the aqueousext'racting solutions ywere .variedand in the rst test .anaqueous -sodiumhydroxidefsolution alone VWasemployed.

.The .percentages "by .Weight of' protein "nitrogen and carbohydrate. inthe isolatedprotein 'are indilcated'. in columns`2` and?. respectively,andjwere obtained by [applying conventional analytical rnetl'iod'sto.vtheisolated protein. `Itwill'lbe ob- Y served. thatv aslthe.conce'rltra'.'tion fof'calcium chloridelis. increased' upto'.0.61'N. While'maintaining vthe..concentration. .of vtheso'diumhydroxide at ab'oui'flllt'I N.r the amount of. canbohydrate remaining inthe product is decreased. ".Thequalitypf .thefisolated protein .withregardtocolor is indi'- 4cated thelastfthree. columns Yof' Tablefl. The.pigmentation was determinedlby the Percentage of.-

-.transmittance ofi .a 1` solution of .Lh'e .isolated proteininanfaqueouslOLN. sodium.: hyadroxide-ysolution -a.t,-420,`.520, and690m wave bands. .It is; lto ber vobserved .that A.as the calciumchloride.l concentration is increased 'up to 0.6 N. .theg` percentage.-of lightetransmission, .that is, the qualityicfl the.productwith-regard.tofcolor.. is. also increased.. Likewise, .where.flaxseed.v meal-.isuSed alsraestartingfmateriaL Vthe percentage-v. ofprotein nitrogen in the productr-isj.also increased up .to the i'.concentration of. 0.6 NQ. calciumchloride. f'lihisrindieatesthat.- a;purer protein .may .be .ex- .tracted .-ffromn flaxseed`.mealby.employing.l the. instant; novelvprocess.. instead .of usingmerely an aqueous solution of sodium hydroxide.

;."l'1o.a.boutl.l00. fgrams.of solventlextracte'diiiely .ground-soybean.meal was c.slc'ilv'vlyf added". with v.tf-igoronsl mechanical..fsti'rririf'g4 approximately 1600 cc. 'of an aqueous lsolutionV ofabout 05"`N.

obtained 'when about a 015 N. aqueous .'sodiuh'cl hydroxide: solutionisemployed lline B indicates the Yresults `obtained r'Whenaqueous'.'solution of about 0.5 N. sodiumv -hydroxide containing about-0154 N. equivalentsof calcium chloride is employed;

V'Thegdatasjet forth in Table 'II was obtained by analysis' 'of the'protein isolated in accordance vvith:A the procedure sletA forth inExample' II'. "The proteinsv'were Visolatedfromj severaly .oilseed mealsas 'indicated in column v1. :.Inl teach' .'case, prod? nets-wereobtained by. employinglaf05" N'. vs odiujin vliydroxiole aqueoussolution 'anda' 0.5 N. "calcium chlorideOi-S N. sodium hydroxide aqueoussolution. Eachproduct Lthus obtainedA was "subjected to thesameanalytical' procedures usedi'in' obtaining the data recorded-inrI'vable I.

That there is an improved quality of the isolated protein obtained by'the use of the instant novel `vprocessr'over that `obtainedby the .useof aqueousO-.i N. sodium hydroxide is evident. In all cases more proteinnitrogen ispresentin the protein isolated by means of theca1cium.chloride' 'sodium v'hydroxide solution-vthanby -merely anaqueousso'dium hydroxidessolution. It should-be fnoted that a 2 to 3%higher nitrogen-content represents considerable improvement in proteinquality since each .gram of protein'nitrogenvcor- `sodiumhydroxidesolution.. lThese oilseedf` meals decreasein carbohydrate content from5.0% and 7 7.7%; respectively, to belowj 1 The proteins f-romcottonseedand soybean meals decrease'in carbohydrate contentfrom 4.8% and v4.2% to2.7%'.'in either case, but the castor bean protein from their.respective. o ilseed meals in accordance withthe procedure vasset; forthinfExam-ple II. ,The proteins .thusA .tslelatedY were then. varlaIyZd-for glutamic acid, methionine, and cystine.

' Table II Protein Percent Lgitl mi ance o .ercen 11; Ouseed Mea] UsedPercent By Pellthgy solo. in 0.5 N. NaOH f 'Weight Carbhy at- Nitrogenrate v 420mg 520m 690mg Castorbeens: i Y i -A .3.;.--7 y 12.77- v1 1.7T4-1.9; 76.0 89.5 13..-.- 14. 63V 1. 7 65. 8 90. 5 96. 5 Soybeans: f Y

12.83 4.8 2.2 '5.4' 43.9 l5. 56 2. 7 9. 8 34. 7 72. 2 *Y 11.35 7.7 7.1"'29.0 77.0" 17.10 0.9 59.1 91.7 Y 946.0

All values are corrected for moisture and ash.

contain '1.7% carbohydrates Whether treated with a sodium hydroxide orwith a calcium chlo-y ride-sodium hydroxide solution.

In all cases the final product was found to have'- benefitted withregard to color removal when a calcium chloride-sodium hydroxidesolution was employed instead of an aqueous sodium hydroxide solution.However, each protein benets to a differentextent. Improvements in theappearance of cottonsee'd and soybean proteins are'rela.- tively'slight; in z.castor bean and peanut Droteins. the improvement is j muchmore Vvappre- The results recorded in Table III clearly indicate thatall of the calcium chloride-sodium hydroxide extracted proteins arericher in glutamic acid than the sodium hydroxide extractedproteins.

The methionine concentrationsof the proteins isolated fromsodiumfhydroxide extractions are quite uniform for all of the oilseedsranging betweeri'0.7%V and" 1.0% bylweight. i'Ih'ose concentrationsobtained from the calcium" chloridesodium hydroxide extractions are,uwith the exyluti'on:comprising essentially 0.5 N. sodium hydroxideand 0.5 N. calcium chloride are set forth. Line'A of Table III indicatesthe results obtained kWhenabout a 0.5 N. aqueous sodium hydroxidesolution is employed; line B indicates the results obta'ined'when anaqueous solution of about 0.5 N." 'sodium hydroxide containing about 0.5N. equivalents of calciumchloride is employed.

Table II y Percent l Percent Percent Oilseed Meal Used Bggaelllt ByWeight By Weight Add Methionine Cystine 10. 7 Y 0. 9 OS2 14. 5 1. 1 0-915. 2 0. 8 0.15 19. 2 o. s 0.9 15. 5 o. 7 o. 5 20. 1 1. 2 o. 5 12. 9Y1'. o o. 5 `17.0 0.8 OQ "11. 7 o. 7 vo 18. 8 1. 0 1. 1

' All values'are corrected for moisture and ash. l i f ''The procedurefollowed in obtaining the data ception of soybeanand cottonseed meals;slightly higher.

The cystine values indicated vin column 4v of Table III clearly indicatethe destructiveaction of alkali on cystine-,containi'ng proteins. AExcept for peanut Ym'eal, fa.ll"`offtl'1e'sodium hydroxide lextractedproteins contain very much less than the calcium' chloride-sodium,hydroxide extracted proteins. 'This is particularly evident in the'case`o-fflaxseed Vmeal Where not a trace of remaining cystine could bedetected inthe sodium hydroxide extracted protein, yet 1.1 was found intheprep'- aration isolatedfrom' calcium chloride-sodium hydroxideextracts. Y "f Table IV summarizes' the yields obtained by employingthe'insta'ntmovel process as set rforth in Example II. Table'IV, line` AindicatesV the results obtained when about va'0`.5 N. aqueous sodiumhydroxide solution" is employed; lin'e'lv indicates the results obtainedWhen an aqueous solution of about 0.5 N. sodiumhydroxide containingabout G25 N. equivalents of calcium chloride is employed.

As in the previoustables, a comparison/is made between the resultsobtained by employing calcium chloride-sodium hydroxideextractingvs'olulon and the results obtained-by employing k'merelysodium" hydroxide" solution.l

' "Column 1 of Table IV indicates the type ofoiltnaxxtsilcsstotakmealanitrggmenffrmth. .fflveftypesoff, seedairreali. tested columng,.iff/.heefresiutse- Howeverz..

{tnt-...analysis ture, separating solid material therefrom, adding;Vv

to.-the; .resultingsolutionv sufficient mineral acid;

nonoxidizing: under the conditions obtained, .to adjust the pH of .th-e`aforesaid solution-Lto; be-f:

, tracted by the instant novel process if soybean,

axseed, or peanut meal is employed. The protein nitrogen referred to isthat capable of precipitation by the proper pH adjustment as indicatedin column 4. From these data, it is indicated that, quantitatively,castor beans appear to be the best source when'a sodium hydroxidesolution is the solvent, but peanut meal is superior quantitatively whencalcium chloride-sodium hydroxide extracts are made. It is to be notedthat these data referring to percentages of extractedl and recoverednitrogen are of value only to the extent that they reveal comparativeefficiencies of two or more solvents. The absolute amounts of proteinrecovered depend also upon the nitrogen content of the startingmaterial. This is recorded in column 2 of Table IV.

The data in Table IV are primarily concerned with a. comparison ofyields when the two solvents are employed. This table does not indicatethe quality of the protein produced. For example, the yield of flaxseedmeal, as indicated in column 6 of TableIV, isv only 12.4. grams when acalcium chloride-sodium hydroxide solution is employed, as compared with23.9 grams if extracted with a sodium hydroxide solution. However,reference to Tables I and II indicates a marked improvement in thequality of vthe calcium chloride-sodium hydroxide extracted protein bothin the case of flaxseed and the other vegetable seed meals. Theeconomical production of a protein of such a high d-egree of purity asis accomplished by the instant novel process compensates for the loweryield which is generally obtained. Y

Having fully described the nature and character of the invention, whatis desired to be secured by Letters Patent is:

1. A process for extracting the proteinaceous matter from oleaginousproteinaceous vegetable residue which comprises admixing said residuefrom which substantial amounts of oil have been previously removed withan aqueous mixture prepared by adding to an aqueous calcium chloridesolution sufficient aqueous sodium hydroxide solution to obtain aconcentration of about 0.5 N. sodium hydroxide and between about 0.1 andabout 0.7 N. calcium chloride in the combined mixture, vigorouslyagitating the resulting mixtween about 3.5 and about 4.5, and separatingthe precipitated proteinaceous matter therefrom.

2. A process according to claim 1 wherein the vegetable residuecomprises essentially flaxseed meal from which substantial amounts ofoilhave been removed.

3. A process for extracting proteinaceous matter from oleaginousproteinaceous vegetable residue which comprises admixing said residuefrom which substantial amounts of oil have been previously removed withan aqueous solution prepared by adding with agitation to an aqueouscalcium chloride solution sunlcient aqueous sodium hydroxide solution toobtain a concentration of about 0.5 N. sodium hydroxide and betweenabout 0.1 and about 0.7 calcium chloride in the combined mixture,vigorously agitating the resulting mixture, separating the solidmaterial therefrom, adding to the resulting solution suflicient mineralacid, nonoxidizing under the conditions obtained, to adjust the pH ofthe aforesaid solution to between about 3.5 and about 4.5, andseparating the precipitated proteinaceous matter therefrom.

4. A process according to claim 3 in which the vegetable residue isobtained from the oil extracted meal of flaxseed and wherein the nal pHadjustment is to about 3.8.

5. A process according to claim 3 in which the vegetable residue isobtained from the oil extracted meal of soybeans and wherein the nal pHadjustment is to about 4.1.

6. A process according to claim 3 in which the vegetable residue isobtained from the oil extracted meal of cottonseedand wherein the nal pHadjustment is to about 3.8.

7. A process for extracting proteinaceous matter from oleaginousproteinaceous vegetableresidue which comprises admixing said residuefrom which substantial amounts of oil have been previously removed withan aqueous mixture prepared by adding to an aqueous calcium chloridesolution having a concentration of between about 0.2 and about 1.4 N.about an equal volume of a solution of about 1.0 N. sodium hydroxide,vigorously agitating the resulting mixture, separating the solid mattertherefrom, adding tothe resulting solution sufficient mineral acid,nonoxidizing of lthe aforesaidsolution to between about 3.5 and .about4L5f ands'eparating proteinaceous matter therefrom.v Y f i 8. A processfor extracting proteinaceous matter from oleaginous proteinaceousvegetable residue which comprises admixing said residue from whichsubstantial amounts of oil have been previously removed with an aqueousmixture pre-lv pared by adding with agitation to an aqueous calciumchloride solution having a concentration of `between about 0.2 and about1.4 N. about anequal volume of a, solution of about 1.0 N. sodium lhydroxide, vigorously agitating the resulting mixture for about 2hours,rseparating the solid matter therefrom, adding to the resultingsolution sufficient mineral acid, nonoxidizing under the conditionsobtained, to adjust the pH of the aforesaid solution to between about3.5 and about 4.5, and separating the precipitated proteinaceous mattertherefrom.

9. A process for extracting proteinaceous matthe precipitated 12ter-from substantially oil free mucilage-contain-ff ing axseed mealwhich comprises admixing'said-' meal and an aqueous mixturepreparedbyadd- 'f ing with agitation 'to an 'laqueous 'calcium chlo'-'vride solution havinga concentration of between about 0.2 and about 1.4N. about an equal volume s' of about 1.0 N. sodium hydroxide, agitatingthe f resulting mixture vigorously for about 2 hours,

separating solid material therefrom,acidifying 10' thejresultingsolution with concentratednhydro# chloric acid to a pH` of between about3.7 Vand about 3.8, and separating theVA precipitated proteinaceousmatter therefrom. I

REFERENCES Ci'rED smith et a1.; 1nd. and Eng. chem., voi.V so;

1. A PROCESS FOR EXTRACTING THE PROTEINACEOUS MATTER FROM OLEAGINOUSPROTEINACEOUS VEGETABLE RESIDUE WHICH COMPRISES ADMIXING SAID RESIDUEFROM WHICH SUBSTANTIAL AMOUNTS OF OIL HAVE BEEN PREVIOUSLY REMOVED WITHAN AQUEOUS MIXTURE PREPARED BY ADDING TO AN AQUEOUS CALCIUM CHLORIDESOLUTION SUFFICIENT AQUEOUS SODIUM HYDROXIDE SOLUTION TO OBTAIN ACONCENTRATION OF ABOUT 0.5 N. SODIUM HYDROXIDE AND BETWEEN ABOUT 0.1 ANDABOUT 0.7 N. CALCIUM CHLORIDE IN THE COMBINED MIXTURE, VIGOROUSLYAGITATING THE RESULTING MIXTURE, SEPARATING SOLID MATERIAL THEREFROM,ADDING TO THE RESULTING SOLUTION SUFFICIENT MINERAL ACID NONOXIDIZINGUNDER THE CONDITIONS OBTAINED, TO ADJUST THE PH OF THE AFORESAIDSOLUTION TO BETWEEN ABOUT 3.5 AND ABOUT 4.5, AND SEPARATING THEPRECIPITATED PROTEINACEOUS MATTER THEREFROM.